EP1280563B1 - Medizinische implantat-materialien - Google Patents

Medizinische implantat-materialien Download PDF

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EP1280563B1
EP1280563B1 EP01925694A EP01925694A EP1280563B1 EP 1280563 B1 EP1280563 B1 EP 1280563B1 EP 01925694 A EP01925694 A EP 01925694A EP 01925694 A EP01925694 A EP 01925694A EP 1280563 B1 EP1280563 B1 EP 1280563B1
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Prior art keywords
transglutaminase
polymer
fibronectin
poly
pcl
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French (fr)
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EP1280563A1 (de
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Martin Griffin
Deborah Heath
Paul Christian
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Aston University
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Aston University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24

Definitions

  • the present invention relates to materials for use in medicine, in particular medical implant materials.
  • the invention further provides a method of improving the biocompatibility of a medical implant material.
  • biomaterials are now used in the repair of damaged tissue (e.g. bone and skin), in prosthetic devices such as artificial hip and knee joints, heart valves, and blood vessels, and in drug delivery devices.
  • damaged tissue e.g. bone and skin
  • prosthetic devices such as artificial hip and knee joints, heart valves, and blood vessels
  • drug delivery devices e.g., drug delivery devices
  • PCL poly( ⁇ -caprolactone)
  • PCL is a semi-crystalline linear resorbable synthetic aliphatic polyester, - (-O-(CH 2 ) 5 -CO-)-.
  • the polymer When implanted in vivo, the polymer is readily degraded non-specifically by hydrolytic enzymes, esterases and carboxypeptidases. Pitt et al. (1981), showed that degradation of PCL in vivo and in vitro proceeds via hydrolytic chain scission of ester linkages until the segments are sufficiently small to diffuse through the polymer bulk. Once the polymer reaches the molecular weight of 5000 Daltons, significant weight loss is observed, which is dependent on particle size. Chain scission has also been shown to be associated with an increase in crystallinity, which partially determines the rate of degradation (see review by Smith et al., 1990).
  • PCL tricarboxylic acid
  • TCA tricarboxylic acid
  • PCL Poly(L-Lactide)
  • PLA Poly(L-Lactide)
  • PLA is one of the strongest polyesters and has a resorption time of greater than one year, probably in the range of 2-3 years. This would be advantageous, for example, in a 3-D tissue construct/scaffold, where the implant resorption rate needs to be adjusted according to the tissue repair rate.
  • PCL is a plastic at body temperature. Its mechanical properties make it ideal for drug delivery systems, but not for the internal fixation of bone.
  • Lowry et al., (1997) made reinforced PCL with phosphate glass fibres in the form of intra-medullary pins for the internal fixation of bone. This study was performed in the rabbit model and histological evidence showed that the composite was well tolerated, with minimal inflammation around the pin.
  • the review by Daniels et al. (1990) illustrates that the mechanical properties of polymers and composites can be improved by reinforcing the materials with alumina, alumina-boria-silica, calcium metaphosphate glass fibers and carbon.
  • PCL can also be blended with natural polymers, e.g . collagen, fibronectin, hyaluronic acid and glycosaminoglycans. These natural polymers are all part of the extracellular matrix (ECM) that cells produce and secrete. It is thought that by incorporating natural polymers with synthetic polymers, osteoconductance and biocompatibility properties could be combined with the physical and mechanical properties of the synthetic component, making bioartificial polymers a good bioactive biomaterial substitute.
  • ECM extracellular matrix
  • Giusti et al., (1994) discuss the importance of blending collagen with polymeric materials for use as medical devices and show how blending also increases the mechanical and thermal properties as compared to the individual components.
  • Cascone et al., (1994) demonstrated the use of collagen and hyaluronic acid based polymeric bioartificial polymers as a successful drug delivery system for the release of growth hormone.
  • the number of proteins acting as glutaminyl substrates for transglutaminases is highly restricted since both the primary structure and conformation are critical.
  • the only requirement of the acyl-acceptor substrate is the presence of a suitable primary amine, e.g. the ⁇ -amino group of peptide bound lysine residues and small primary amines.
  • a suitable primary amine e.g. the ⁇ -amino group of peptide bound lysine residues and small primary amines.
  • Different types of transglutaminase enzyme differ in their specificity for a given glutaminyl substrate.
  • the plasma transglutaminase blood coagulation factor XIIIa acts on a limited range of glutaminyl substrates compared to tissue (or type II) transglutaminase (tTG).
  • tTG also binds GTP and GDP, which is thought to be important in its regulation by Ca 2+ (see Smethurst and Griffin, 1996).
  • a further key difference between the types of transglutaminase is in their distribution.
  • tTG has been mainly described as a cytosolic enzyme and does not contain a typical hydrophobic leader sequence for secretion, it may be found both in the cytosol and membrane associated depending on the cell type.
  • the biological function of tTG has yet to be determined.
  • tTG can act at the cell surface, facilitating cell adhesion (Borge et al., 1996) and cell spreading (Jones et al., 1997) and the modification of the extracellular matrix (ECM) (Aeschlimann et al., 1995, Barsigian et al., 1991, Bowness et al., 1988 and Bendixen et al., 1993).
  • ECM extracellular matrix
  • transglutaminase enzymes to cross-link proteins has been exploited in the development of biological glues for promoting adhesion between tissue surfaces.
  • biological adhesive compositions comprising a tissue transglutaminase are disclosed in WO 94/28949. These compositions also comprise a divalent metal ion co-factor, which plays a regulatory role in the functional activity of transglutaminase enzymes (see Casadio et al., 1999, Eur. J. Biochem. 262 , 672-679).
  • the use of a transglutaminase as a biological adhesive, wherein the enzyme is used in an active form is also disclosed in EP 0 530 804 A (and references cited therein).
  • a first aspect of the invention provides a medical implant material comprising a mammalian transglutaminase and a polymer, wherein the transglutaminase is provided in the absence of free divalent metal ions and wherein the polymer is associated with a binding protein for binding the transglutaminase.
  • 'medical implant material we include a material for implantation into the human or animal body, such as a material for use as an artificial tissue (e.g. bone, teeth and skin), prosthetic devices (e.g. joints, heart valves, blood vessels) and drug delivery devices.
  • an artificial tissue e.g. bone, teeth and skin
  • prosthetic devices e.g. joints, heart valves, blood vessels
  • transglutaminases prepared (i.e . extracted) from mammalian tissue samples, as well as mammalian transglutaminases expressed by recombinant means.
  • transglutaminases prepared ( i.e . extracted) from mammalian tissue samples, as well as mammalian transglutaminases expressed by recombinant means.
  • mammalian transglutaminases expressed by recombinant means.
  • 'free divalent metal ions we mean unchelated divalent metal ions, such as Ca 2+ ions, which are available to interact with the transglutaminase and regulate the functional activity of the enzyme.
  • transglutaminases which are provided in the presence of divalent metal ions that are bound to a chelating agent, and hence are not available to interact with the enzyme.
  • the transglutaminase is a tissue transglutaminase.
  • the transglutaminase may be obtained from a culture of human cells that express a mammalian transglutaminase, using cell culture methodology well known in the art.
  • Preferred cell line sources of such transglutaminases include human endothelial cell line ECV304 (for tissue transglutaminase) and human osteosarcoma cell line MG63.
  • the source of the transglutaminase may be selected according to the particular use (e.g. site of implantation) of the medical implant material.
  • the medical implant material is to be used as artificial bone, it may be beneficial for the material to comprise a bone-derived transglutaminase.
  • the nucleic acid molecule e.g. cDNA, encoding the transglutaminase may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • Baculovirus expression system in insect cells may be used (see Richardson et al., 1995), Methods in Molecular Biology Vol 39, J Walker ed., Humana Press, Totowa, NJ).
  • the expression constructs will desirably also contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. (Hastings et al, International Patent No. WO 98/16643, published 23 April 1998)
  • the recombinant transglutaminase can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • Examples of an inducible mammalian expression vectors include pMSG, also available from Pharmacia (Piscataway, NJ, USA), and the tetracycline (tet) regulatable system, available form Clontech.
  • the pMSG vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • the tet regulatable system uses the presence or absence of tetracycline to induce protein expression via the tet-controlled transcriptional activator.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or transladonal controls.
  • One such method involves ligation via homopolymer tails.
  • Homopolymer polydA (or polydC) tails are added to exposed 3' OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases.
  • the fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined by DNA ligase.
  • Another method involves ligation via cohesive ends.
  • Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
  • the variant may have increased ability to facilitate the colonisation of medical implants by cells, wherein said increased ability is independent of the enzyme activity of the variant but is related to some other property of the variant.
  • have increased ability of the variant transglutaminse to facilitate the colonisation of medical implants by cells may be associated with an increased ability to bind endogenous ( i.e . host) proteins such as receptors.
  • the enzyme activity of variant mammalian transglutaminases may be measured by the biotin-cadaverine assay, as described in the Examples and as published in (Jones et al., 1997, J. Cell. Sci. 110 , 2461-2472).
  • tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin.
  • transglutaminase activity may be measured by the incorporation of [ 14 C]-putrescine incorporation into N,N'-dimethylcasein, as outlined by Lorand et al., 1972, Analytical Biochemistry 50 , 623.
  • the increased ability of the variant enzyme to facilitate the adhesion and spreading of cells on medical implants may be measured by the methods disclosed herein.
  • Other synthetic polymers include methacrylates poly(ethylmethacrylate), ethylacrylate, tetrahydrofurfurylmethacrylate, hydroxyethylmethacrylate, silastic, poly(tetrafluoroethylene), medpore (porous polyethylene), poly(orthoester), and poly(dioxane).
  • the medical implant material comprises poly-( ⁇ -caprolactone).
  • a 'binding protein' we include a protein or polypeptide which is able to bind to a mammalian transglutaminase, i.e. a transglutaminase binding protein.
  • the transglutaminase binding protein binds to the mammalian transglutaminase with a binding affinity greater than 10 3 L/mol. More preferably, the affinity constant is greater than 10 4 L/mol, for example greater than 10 5 L/mol, 10 6 L/mol, 10 7 L/mol, 10 8 L/mol, 10 9 L/mol, 10 10 L/mol, or 10 11 L/mol,
  • the transglutaminase binding protein is fibronectin or a fragment thereof that is capable of binding to a transglutaminase.
  • the polymer may be incubated overnight at 4°C in a solution comprising a transglutaminase binding protein.
  • a transglutaminase binding protein may be immobilised on the polymer surface by evaporation or by incubation at room temperature.
  • the transglutaminase binding protein is covalently linked to the polymer, e.g . at the external surface of the polymer.
  • the transglutaminase is provided in the absence of free Ca 2+ ions.
  • the reinforcing agent may be any substantially non-toxic material that can be blended or mixed with the polymer/transglutaminase components of the medical implant material to increase its strength.
  • biocompatibility we mean the ability of the medical implant material to facilitate its colonisation by host cells aud to enhance proliferation of host cells therein.
  • biocompatibility is not intended to cover mere adhesion of host cells to the medical implant material, but rather relates to an interaction between the host cells and implant materials which permits colonisation to occur.
  • biocompatibility includes the ability of said material to support cell attachment, cell spreading, cell proliferation and differentiation.
  • Biocompatibility of a medical implant material may be assessed using methods known in the art (see Examples). For example, increased biocompatibility of a medical implant material is associated with an increase in the ability of the material to facilitate cell attachment, cell spreading, cell proliferation and differentiation. In addition, the material should not induce any substantial loss in cell viability, i.e. via the induction of cell death through either apoptosis or necrosis.
  • the differentiation of a cell type is measured in different ways depending on the cell type in question. For example, for osteoblasts cells in culture, alkaline phosphate together with extracellular matrix (ECM) deposition, e.g . collagen 1, fibronectin, osteonectin and osteopontin, can be used as a marker.
  • ECM extracellular matrix
  • the ability of cells to proliferate and deposit ECM is important to any material that is to be used as an implant, this includes endothelial cells, chondrocytes and epithelial cells etc.
  • a further preferred embodiment of the second aspect of the invention provides the use of a mammalian transglutaminase to improve the biocompatibility of a medical implant material further comprising one or more additional polymer(s).
  • the medical implant material may be a co-polymer or a blend of polymers.
  • the medical implant material is artificial bone.
  • PCL Poly( ⁇ -caprolactone) (PCL650; Solvay Interox) was purchased in pellet form and pressed in a square cavity at a temperature above melting point, in order to form a 4mm thick sheet. PCL discs (6 mm in diameter) were then stamped from the polymer sheet using a cork borer and sterilised under UV light for 1 hour each side prior to use.
  • HOB cells were grown on the PCL in 10% serum containing DMEM to initially assess the interaction of cells with this particular polymer surface.
  • the toluidine blue stain allowed the attachment, spreading and proliferation of the cells on the PCL to be observed.
  • T.E.M. allowed the ultra structure of the cells to be observed on the biomaterial. Both these techniques would indicate whether the polymer is biocompatible with this particular cell type.
  • PCL discs were placed in a 48-well plate (3 replicates per time point) and HOB cells were seeded onto the biomaterial in 10% serum containing DMEM at a density of 1.7x10 5 /cm 2 . Thermanox was used as a positive control surface. The plate was then incubated at 37°C in 5% CO 2 . At days 1, 2, 4 and 8 days after cell seeding, the medium was removed and the cells were washed in sterile PBS. The samples were fixed overnight in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) then washed in the same buffer and secondary fixed using 1% osmium tetroxide in Millonig's buffer for 1 hour.
  • PCL discs were coated with different concentrations of fibronectin (0 to 50 ⁇ g/ml) by the evaporation technique described above.
  • the fibronectin was detected using the fibronectin ELISA assay (Gaudry, 1998).
  • the polymers were initially washed in 3 x 100 ⁇ l of PBS and then blocked with 100 ⁇ l of 3% w/v bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • the plate was incubated for 1 hour at room temperature and then washed with 3 x 100 ⁇ l of PBS before the addition of 100 ⁇ l of the primary antibody (polyclonal rabbit anti-human plasma fibronectin, diluted 1:5000 in blocking buffer).
  • PCL discs were coated with 60 ⁇ l of 30 ⁇ g/ml fibronectin in sterile distilled water and then different concentrations of tTG (0-50 ⁇ g/ml) using the two different methods as described above.
  • the discs were washed in 3 x 100 ⁇ l of PBS and then blocked with 100 ⁇ l of 3 % w/v BSA in PBS for 1 hour at room temperature.
  • the PCL discs were rinsed with 3 x 100 ⁇ l of PBS and bound tTG was detected using 100 ⁇ l of anti-monoclonal antibody CUB7402 (diluted 1:1000 in blocking buffer).
  • the PCL or TCP were initially blocked with 100 ⁇ l of 3% BSA in 0.1M Tris-HCl (pH 7.4.) at room temperature for 1 hour and then washed with 3 x 100 ⁇ l of 0.1M Tris-HCl.
  • 100 ⁇ l of the following solution was added to the fibronectin coated TCP and PCL: 20 ⁇ g/ml guinea pig tTG, 5mM CaCl 2 , 3.85mM DTT and 0.4% biotin-cadaverine in homogenising buffer.
  • Alkaline Phosphatase (ALP) Assay (Granutest kit obtainable form Merck)
  • the T.E.M. results also indicated that the HOB cells proliferate on the PCL surface as shown by multilayer formation over time.
  • the ultra structure of HOB cells on PCL was seen to be normal. However at 8 days of culture in DMEM containing 10% foetal calf serum, it was found that very few cells remained on the surface. It was thought that the cells must have been removed during the washing processes.
  • the ELISA technique clearly demonstrated the binding of fibronectin to PCL (see Figure 5).
  • the concentration of bound fibronectin increased proportionally up to 50 ⁇ g/ml.
  • the modified ELISA technique also demonstrated the binding of tTG to fibronectin coated PCL when tTG was immobilised by either evaporation overnight or by incubation for 1 h at room temperature (see figures 6 and 7 respectively).
  • Figures 6 and 7 clearly show that tTG can be immobilised to PCL using both methods.
  • the maximum concentration of tTG that can be immobilised to the fibronectin coated PCL is approximately 30 ⁇ g/ml for both immobilisation techniques and the half maximum binding capacity is approximately 15 ⁇ g/ml.
  • Figures 19-21 show the degree of spreading 3 hours after seeding the HOB cells in serum free medium.
  • the cells clearly remained rounded in morphology on the PCL.
  • a lot of cells had detached from the biomaterial surface illustrating that HOB cells require adhesion proteins to spread on PCL.
  • the cells were flat in morphology and had formed a monolayer.
  • the ELISA techniques demonstrated the presence of fibronectin on PCL when immobilised by evaporation and also demonstrated the presence of tTG on fibronectin coated PCL when immobilised by either evaporation overnight or by incubation for 1h at room temperature.

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  • Health & Medical Sciences (AREA)
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  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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Claims (68)

  1. Medizinisches Implantationsmaterial, das eine Transglutaminase von einem Säugetier oder Menschen und ein Polymer umfasst, bei dem die Transglutaminase in Abwesenheit von freien divalenten Metall-Ionen vorgesehen ist und bei dem das Polymer einem Bindungsprotein zum Binden der Transglutaminase zugeordnet ist.
  2. Material nach Anspruch 1, bei dem die Transglutaminase eine Gewebe-Transglutaminase ist.
  3. Material nach Anspruch 1, bei dem die Transglutaminase eine Plasma-Transglutaminase ist.
  4. Material nach einem der Ansprüche 1 bis 3, bei dem die Transglutaminase aus Gewebe oder Zellen von Säugetieren oder Menschen zubereitet ist.
  5. Material nach Anspruch 4, bei dem die Transglutaminase aus menschlichem Gewebe oder menschlichen Zellen zubereitet ist.
  6. Material nach einem der vorhergehenden Ansprüche, bei dem die Transglutaminase eine rekombinante Transglutaminase ist.
  7. Material nach einem der vorhergehenden Ansprüche, bei dem die Transglutaminase eine variante Transglutaminase ist.
  8. Material nach einem der vorhergehenden Ansprüche, bei dem das Polymer ein natürlicherweise auftretendes Polymer ist.
  9. Material nach Anspruch 8, bei dem das natürlicherweise auftretende Polymer ein natürlicherweise auftretendes extra-zelluläres Matrix-Molekül, vorzugsweise Collagen, Fibronectin, Fibrin, Fibrillin, ein Glukosoaminoglykan oder Hyaluronsäure ist.
  10. Material nach einem der vorhergehenden Ansprüche, bei dem das Polymer ein synthetisches Polymer ist.
  11. Material nach Anspruch 10, bei dem das synthetische Polymer aus der Gruppe ausgewählt ist, die Poly-Caprolacton (PCL), Poly-L-Lactid (PLA), Poly-Glykolid (PGA), Poly-DL-Lactid-Coglykolid) (PLG), Methacrylat-Poly-Ethylmethacrylat, Ethylacrylat, Tetrahydrofurfurylmethacrylat, Hydroxyethylmethacrylat, Silicongummi, Poly-Tetrafluorethylen, Medpor (poröses Polyethylen), Poly-Orthoester, Poly-Dioxan und Co-Polymere und Mischungen hiervon umfasst.
  12. Material nach Anspruch 10 oder 11, bei dem das Polymer Poly-e-Caprolacton ist.
  13. Material nach einem der vorhergehenden Ansprüche, bei dem das medizinische Implantationsmaterial ein Polymer umfasst, das mit einem Transglutaminase-Binde-Protein beschichtet, imprägniert, kovalent gebunden oder diesem auf andere Weise beigemischt ist.
  14. Material nach einem der Ansprüche 1 bis 13, bei dem das Polymer mit dem Transglutaminase-Binde-Protein beschichtet ist.
  15. Material nach einem der Ansprüche 1 bis 13, bei dem das Polymer mit dem Transglutaminase-Binde-Protein imprägniert ist.
  16. Material nach einem der Ansprüche 1 bis 15, bei dem das Polymer kovalent an das Transglutaminase-Binde-Protein gebunden ist.
  17. Material nach einem der Ansprüche 1 bis 16, bei dem das Transglutaminase-Binde-Protein ein Transglutaminase-Substrat ist.
  18. Material nach einem der Ansprüche 1 bis 17, bei dem das Transglutaminase-Binde-Protein aus der Gruppe ausgewählt ist, die Fibronectin, Fibrin, Fibrinogen, Collagen, Entactin, Osteonectin, Osteopontin, Thrombospondin, Vitronectin, β-Lactoglobulin und Kasein umfasst.
  19. Material nach einem der Ansprüche 1 bis 18, bei dem das Transglutaminase-Binde-Protein Fibronectin oder ein Fragment hiervon ist, das in der Lage ist, sich an eine Transglutaminase zu binden.
  20. Material nach Anspruch 19, bei dem das Fibronectin-Fragment Aminosäuren 32-608 des N-Terminus von Fibronectin oder ein Fragment hiervon umfasst.
  21. Material nach einem der Ansprüche 1 bis 18, bei dem das Transglutaminase-Binde-Protein Fibrin oder ein Fragment hiervon ist, das in der Lage ist, sich an eine Transglutaminase zu binden.
  22. Material nach einem der vorhergehenden Ansprüche, bei dem das medizinische Implantationsmaterial weiterhin ein Verstärkungsmittel umfasst.
  23. Material nach Anspruch 22, bei dem das Verstärkungsmittel aus einer Gruppe ausgewählt ist, die Aluminium, Aluminiumoxid-Boroxid-Siliziumoxid, Kalzium-Methaphosphat-Glasfasern, Titan und Kohlenstoff umfasst.
  24. Material nach einem der vorhergehenden Ansprüche, bei dem das medizinische Implantationsmaterial weiterhin wenigstens ein zusätzliches Polymer umfasst.
  25. Material nach Anspruch 24, bei dem das wenigstens eine zusätzliche Polymer ein synthetisches Polymer ist.
  26. Material nach Anspruch 24, bei dem das wenigstens eine zusätzliche Polymer ein natürliches Polymer ist.
  27. Material nach Anspruch 24 oder 26, bei dem das wenigstens eine zusätzliche Polymer ein natürlicherweise auftretendes extra-zelluläres Matrix-Molekül, wie z.B. Collagen, Fibronectin, Fibrillin, Fibrin, ein Glukosoaminoglykan oder Hyaluronsäure ist.
  28. Material nach den Ansprüchen 1 bis 27, bei dem das Polymer mit der Transglutaminase beschichtet ist.
  29. Material nach den Ansprüchen 1 bis 27, bei dem das Polymer mit der Transglutaminase imprägniert ist.
  30. Material nach den Ansprüchen 1 bis 29, bei dem die Transglutaminase kovalent mit dem Polymer entweder direkt oder indirekt über das Bindeprotein verknüpft ist.
  31. Material nach den Ansprüchen 1 bis 30, bei dem die Transglutaminase mit einem Gelier-Wirkstoff versehen ist.
  32. Material nach Anspruch 31, bei dem der Gelier-Wirkstoff EDTA oder EGTA ist.
  33. Material nach Anspruch 31 oder 32, bei dem der Gelier-Wirkstoff in einer Konzentration zwischen 5 mM und 0,1 M vorgesehen ist.
  34. Material nach einem der vorhergehenden Ansprüche, bei dem das medizinische Implantationsmaterial künstlicher Knochen ist.
  35. Verfahren zur Verbesserung der Biokompatibilität eines medizinischen Implantationsmaterials, wobei das Verfahren folgende Schritte umfasst:
    (i) Bereitstellen eines medizinischen tmptantationsmateriats, das ein einem Binde-Protein zur Bindung der Transglutaminase zugeordnetes Polymer umfasst und
    (ii) Behandeln dieses Materials mit einer von einem Säugetier oder einem Menschen stammenden Transglutaminase,
    wobei die Transglutaminase in der Abwesenheit von freien divalenten Metall-Ionen vorgesehen ist.
  36. Verfahren nach Anspruch 35, bei dem die Transglutaminase eine Gewebe-Transglutaminase ist.
  37. Verfahren nach Anspruch 35, bei dem die Transglutaminase eine Plasma-Transglutaminase ist.
  38. Verfahren nach einem der Ansprüche 35 bis 37, bei dem die Transglutaminase aus Gewebe oder Zellen von Säugetieren oder Menschen zubereitet ist.
  39. Verfahren nach einem der Ansprüche 38, bei dem die Transglutaminase aus menschlichem Gewebe oder menschlichen Zellen zubereitet ist.
  40. Verfahren nach einem der Ansprüche 35 bis 39, bei dem die Transglutaminase eine rekombinante Transglutaminase ist.
  41. Verfahren nach einem der Ansprüche 35 bis 40, bei dem die Transglutaminase eine variante Transglutaminase ist.
  42. Verfahren nach einem der Ansprüche 35 bis 41, bei dem das Polymer ein natürlicherweise auftretendes Polymer ist.
  43. Verfahren nach Anspruch 42, bei dem das natürlicherweise auftretende Polymer ein natürlicherweise auftretendes extra-zelluläres Matrix-Molekül, vorzugsweise Collagen, Fibronectin, Fibrin, Fibrillin, ein Glukosoaminoglykan oder Hyaluronsäure ist.
  44. Verfahren nach einem der Ansprüche 35 bis 43, bei dem das Polymer ein synthetisches Polymer ist.
  45. Verfahren nach Anspruch 44, bei dem das synthetische Polymer aus der Gruppe ausgewählt ist, die Poly-Caprolacton (PCL), Poly-L-Lactid (PLA), Poly-Glykolid (PGA), Poly-DL-Lactid-Coglykolid) (PLG), Methacrylat-Poly-Ethylmethacrylat, Ethylacrylat, Tetrahydrofurfurylmethacrylat, Hydroxyethylmethacrylat, Silicongummi, Poly-Tetrafluorethylen, Medpor (poröses Polyethylen), Poly-Orthoester, Poly-Dioxan und Co-Polymere und Mischungen hiervon umfasst.
  46. Verfahren nach Anspruch 44 oder 45, bei dem das Polymer Poly-ε-Caprolacton ist.
  47. Verfahren nach einem der Ansprüche 35 bis 46, bei dem das medizinische Implantationsmaterial ein Polymer umfasst, das mit einem Transglutaminase-Binde-Protein beschichtet, imprägniert, kovalent gebunden oder diesem auf andere Weise beigemischt ist.
  48. Verfahren nach einem der Ansprüche 35 bis 47, bei dem das Polymer mit dem Transglutaminase-Binde-Protein beschichtet ist.
  49. Verfahren nach einem der Ansprüche 35 bis 48, bei dem das Polymer mit dem Transglutaminase-Binde-Protein imprägniert ist.
  50. Verfahren nach einem der Ansprüche 35 bis 49, bei dem das Polymer kovalent an das Transglutaminase-Binde-Protein gebunden ist.
  51. Verfahren nach einem der Ansprüche 35 bis 50, bei dem das Transglutaminase-Binde-Protein ein Transglutaminase-Substrat ist.
  52. Verfahren nach einem der Ansprüche 35 bis 51, bei dem das Transglutaminase-Binde-Protein aus der Gruppe ausgewählt ist, die Fibronectin, Fibrin, Fibrinogen, Collagen, Entactin, Osteonectin, Osteopontin, Thrombospondin, Vitronectin, β-Lactoglobulin und Kasein umfasst.
  53. Verfahren nach einem der Ansprüche 35 bis 52, bei dem das Transglutaminase-Binde-Protein Fibronectin oder ein Fragment hiervon ist, das in der Lage ist, sich an eine Transglutaminase zu binden.
  54. Verfahren nach Anspruch 53, bei dem das Fibronectin-Fragment Aminosäuren 32-608 des N-Terminus von Fibronectin oder ein Fragment hiervon umfasst.
  55. Verfahren nach einem der Ansprüche 35 bis 54, bei dem das Transglutaminase-Binde-Protein Fibrin oder ein Fragment hiervon ist, das in der Lage ist, sich an eine Transglutaminase zu binden.
  56. Verfahren nach einem der Ansprüche 35 bis 55, bei dem das medizinische Implantationsmaterial weiterhin ein Verstärkungsmittel umfasst.
  57. Verfahren nach Anspruch 56, bei dem das Verstärkungsmittel aus einer Gruppe ausgewählt ist, die Aluminium, Aluminiumoxid-Boroxid-Siliziumoxid, Kalzium-Methaphosphat-Glasfasern, Titan und Kohlenstoff umfasst.
  58. Verfahren nach einem der Ansprüche 35 bis 57, bei dem das medizinische Implantationsmaterial weiterhin wenigstens ein zusätzliches Polymer umfasst.
  59. Verfahren nach Anspruch 58, bei dem das wenigstens eine zusätzliche Polymer ein synthetisches Polymer ist.
  60. Verfahren nach Anspruch 58, bei dem das wenigstens eine zusätzliche Polymer ein natürliches Polymer ist.
  61. Verfahren nach Anspruch 59 oder 60, bei dem das wenigstens eine zusätzliche Polymer ein natürlicherweise auftretendes extra-zelluläres Matrix-Molekül, wie z.B. Collagen, Fibronectin, Fibrillin, Fibrin, ein Glukosoaminoglykan oder Hyaluronsäure ist.
  62. Verfahren nach einem der Ansprüche 35 bis 61, bei dem der Schritt (ii) das Beschichten des medizinischen Implantationsmaterials mit Transglutaminase umfasst.
  63. Verfahren nach einem der Ansprüche 35 bis 61, bei dem der Schritt (ii) das Imprägnieren des medizinischen Implantationsmaterials mit einer Transglutaminase umfasst.
  64. Verfahren nach einem der Ansprüche 35 bis 61, bei dem der Schritt (ii) das kovalente Verknüpfen der Transglutaminase mit dem medizinischen Implantationsmaterial umfasst.
  65. Verfahren nach einem der Ansprüche 35 bis 64, bei dem der Schritt der Behandlung des medizinischen Implantationsmaterials mit einer Transglutaminase in Anwesenheit eines Gelier-Wirkstoffes durchgeführt wird.
  66. Verfahren nach Anspruch 65, bei dem der Gelier-Wirkstoff EDTA oder EGTA ist.
  67. Verfahren nach Anspruch 65 oder 66, bei dem der Gelier-Wirkstoff in einer Konzentration zwischen 5mM und 0,1 M vorhanden ist.
  68. Verfahren nach einem der Ansprüche 35 bis 67, bei dem das medizinische Implantationsmaterial künstlicher Knochen ist.
EP01925694A 2000-05-12 2001-05-02 Medizinische implantat-materialien Expired - Lifetime EP1280563B1 (de)

Applications Claiming Priority (3)

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GBGB0011356.3A GB0011356D0 (en) 2000-05-12 2000-05-12 Medical implant materials
GB0011356 2000-05-12
PCT/GB2001/001910 WO2001085224A1 (en) 2000-05-12 2001-05-02 Medical implant materials

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EP1280563B1 true EP1280563B1 (de) 2006-12-13

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US7718168B2 (en) 2010-05-18
US20040030408A1 (en) 2004-02-12
GB0011356D0 (en) 2000-06-28
DE60125164D1 (de) 2007-01-25
DE60125164T2 (de) 2007-09-27
AU2001252378A1 (en) 2001-11-20
EP1280563A1 (de) 2003-02-05
ATE347914T1 (de) 2007-01-15
WO2001085224A1 (en) 2001-11-15

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